Pheromone-mediated quorum sensing enables dynamic regulation of cordycepin biosynthesis in Cordyceps militaris

Cordycepin is a nucleoside compound with broad pharmacological activities, whose biosynthesis is tightly regulated by multilayered metabolic mechanisms. Dynamic regulation strategies allow for precise modulation of target product synthesis while maintaining cell growth, thus overcoming the limitations of static control. In this study, a pheromone-mediated quorum sensing (QS) system was reconstructed in Cordyceps militaris to achieve signal-responsive regulation of cordycepin biosynthesis. Phylogenetic and conserved domain analyses revealed that the pheromone receptor PreB and transcription factor SteA of C. militaris share structural homology with Saccharomyces cerevisiae Ste2 and Ste12, indicating potential functional compatibility across species. The synthetic circuit composed of α-pheromone (mfα1) and the pheromone-responsive promoter P_FUS1 exhibited effective signal-responsive activity in C. militaris. Additionally, placing mfα1 expression under the control of the tryptophan-responsive promoter P_ARO9 enabled cell-density-dependent dynamic regulation. This system specifically activated the key biosynthetic genes cns1 and cns2 at high cell density, resulting in a cordycepin titer of 226.5 mg/L, reflecting a 175.9 % increase compared with the wild type. Through 2A-peptide-mediated multigene co-expression and precursor supply engineering (co-expression of cns1 and nt5e), the cordycepin yield was further elevated to 447.9 mg/L, marking a 445.3 % improvement. This work provides a novel dynamic regulation strategy for efficient cordycepin biosynthesis in C. militaris and lays the groundwork for the development of synthetic biology tools in medicinal fungi.